nutrients Article
In Vitro Anticancer Properties of Table Grape Powder Extract (GPE) in Prostate Cancer Avinash Kumar 1 , Melinee D’silva 1 , Kshiti Dholakia 1 and Anait S. Levenson 2, * 1
2
*
Arnold & Marie Schwartz College of Pharmacy and Health Sciences, Long Island University, Brooklyn, NY 11201, USA;
[email protected] (A.K.);
[email protected] (M.D.);
[email protected] (K.D.) College of Veterinary Medicine, Long Island University, Brookville, NY 11548, USA Correspondence:
[email protected]; Tel.: +1-516-299-3692 or +1-718-246-6323
Received: 1 November 2018; Accepted: 15 November 2018; Published: 20 November 2018
Abstract: Although the link between diet and cancer is complex, epidemiological data confirm that diet is a risk factor for prostate cancer and indicate a reduced prostate cancer incidence associated with a diet rich in vegetables and fruits. Because of the known protective effect of grape seed extract (GSE) against prostate cancer, we evaluated the effects of grape powder extract (GPE) on cell viability, proliferation, and metastatic capability. Importantly, we explored the possible novel mechanism of GPE through metastasis-associated protein 1 (MTA1) downregulation in prostate cancer, since our previous studies indicated resveratrol (Res)- and pterostilbene (Pter)-induced MTA1-mediated anticancer activities in prostate cancer. We found that GPE inhibited the cell viability and growth of prostate cancer cells only at high 100 µg/mL concentrations. However, at low 1.5–15 µg/mL concentrations, GPE significantly reduced the colony formation and wound healing capabilities of both DU145 and PC3M cells. Moreover, we found that GPE inhibited MTA1 in a dose-dependent manner in these cells, albeit with considerably less potency than Res and Pter. These results indicate that stilbenes such as Res and Pter specifically and potently inhibit MTA1 and MTA1-associated proteins compared to GPE, which contains low concentrations of Res and mainly consists of other flavonoids and anthocyanidins. Our findings support continued interest in GPE as a chemopreventive and anti-cancer agent against prostate cancer but also emphasize the unique and specific properties of stilbenes on MTA1-mediated anticancer effects on prostate cancer. Keywords: grape powder extract; prostate cancer; MTA1
1. Introduction Despite progresses in understanding the molecular mechanisms of prostate cancer (PCa), it is still the most frequently diagnosed cancer in men in the United States, specifically in recent years, in which life expectancy has increased. Most men acquire PCa during their lifetime because of risk factors such as age and diet. Dietary bioactive polyphenols with anti-inflammatory, antioxidant, and anticancer properties have been of intense interest for use as chemopreventive agents against PCa. Particularly, stilbenes such as resveratrol (trans-3,5,4’-trihydroxystilbene, Res) and its natural analogs including pterostilbene (trans-3,5-dimethoxystilbene, Pter), found in grapes and berries [1,2], have attracted attention as potential pharmacological approaches for primary and clinical chemoprevention of PCa [3–16]. However, only limited studies support separate polyphenol(s) use in human chemoprevention due to their low bioavailability and rapid metabolism [17–19]. Therefore, grape extract, which contains a mix of various polyphenols including stilbenes, might present improved pharmacokinetics and superior pharmacological potency to stilbenes alone and may hold greater potential as a natural Nutrients 2018, 10, 1804; doi:10.3390/nu10111804
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product productdrug. drug.Grape Grapeseed seedextract extract(GSE), (GSE),which whichisisactively activelyavailable availableas asaahealth healthfood foodsupplement, supplement,has has product drug. Grape seed extract (GSE),capabilities whichisisactively actively available as a a health food supplement, has product drug. Grape seed extract (GSE), which available as health food supplement, been shown to have strong antioxidant [20], cardiac benefits [21,22], neurological effects been shown to have strong antioxidant capabilities [20], cardiac benefits [21,22], neurological effects been shown totohave antioxidant capabilities [20], cardiac benefits neurological effects has[23], been shown havestrong strong antioxidant capabilities [20], cardiac benefits [21,22], neurological and cancer preventive and anticancer Particularly, it[21,22], been [23], and cancer preventive and anticanceractivities activities[24–30]. [24–30]. Particularly, ithas has beenshown shownthat thatGSE GSE [23],anticancer andand cancer preventive and anticancer activities [24–30]. Particularly, it has itbeen shown that GSE effects cancer preventive and activities [24–30]. Particularly, has been shown has effects against ininanticancer vitro Importantly, specific signaling has[23], anticancer effects againstPCa PCa vitroand andin invivo vivo[24,28,29,31]. [24,28,29,31]. Importantly, specific signaling has anticancer effects effects against as PCaGSE-regulated, inPCa vitroinand inand vivo Importantly, specific signaling that GSE has anticancer vitro in[24,28,29,31]. vivo [24,28,29,31]. Importantly, specific pathways were including androgen (AR)-mediated pathways were identified identified against as GSE-regulated, including androgen receptor receptor (AR)-mediated pathways were identified as GSE-regulated, including androgen receptor (AR)-mediated signaling pathways were[24] identified as GSE-regulated, including androgen receptor (AR)-mediated transcription ofofgenes and ofofthe ofofextracellular signal-regulated kinase transcription genes [24] andinhibition inhibition theactivation activation extracellular signal-regulated kinase1/2 1/2 transcription of genes [24] and inhibition of the activation of extracellular signal-regulated kinase transcription of genes [24] and inhibition of the[28]. activation of extracellular signal-regulated kinase 1/2 1/2 (ERK associated apoptotic effects (ERK1/2) 1/2)with with associated apoptotic effects [28]. (ERK 1/2) with associated apoptotic effects [28]. (ERK 1/2) with associated apoptotic effects [28]. Our interest includes AR-independent pathways Our interest includes AR-independent pathwaysthat thatplay playaarole roleininthe theprogression progressionofofPCa. PCa.One One Our interest includes AR-independent pathways that play a role in the progression of PCa. One interest includes AR-independent pathways that of play a role in the progression of PCa. One ofand ofofOur these pathways isisrepresented by protein 11(MTA1) these pathways represented byan anoverexpression overexpression ofmetastasis-associated metastasis-associated protein (MTA1) and of pathways these pathways is represented by an overexpression of metastasis-associated protein 1 (MTA1) and these is represented by an overexpression of metastasis-associated protein 1 (MTA1) and the the subsequent activation of MTA1-mediated pro-oncogenic signaling associated with the the subsequent activation of MTA1-mediated pro-oncogenic signaling associated with the the subsequent activation of MTA1-mediated signaling associated withofthe the subsequent activation of MTA1-mediated pro-oncogenicpro-oncogenic signaling associated thedemonstrated progression progression ofof PCa toto metastasis Clinical studies have progression PCa metastasis [3,11–13,16,32–36]. [3,11–13,16,32–36]. Clinical studieswith have demonstrated the progression of[3,11–13,16,32–36]. PCa to metastasis [3,11–13,16,32–36]. Clinical studies have clinicopathological demonstrated PCa to metastasis Clinical studies have tissues demonstrated correlation of high MTA1the correlation ofof high in aggressive correlation high MTA1 MTA1 expression expression in prostate prostate tissues with with the aggressive clinicopathological correlation ofofhigh MTA1 expression in prostate tissues with aggressive clinicopathological expression in prostate tissues with aggressive clinicopathological characteristics ofinin tumors, signifyingwe characteristics signifying MTA1 as target PCa. characteristics oftumors, tumors, signifying MTA1 asaapotential potentialtherapeutic therapeutic target PCa.Therefore, Therefore, we characteristics of tumors, signifying MTA1 as a potential therapeutic target in PCa. Therefore, we MTA1 as a potential therapeutic target in PCa. Therefore, we have intensively investigated andand have haveintensively intensivelyinvestigated investigatedand andreported reportedon onthe theMTA1-mediated MTA1-mediatedanticancer anticancerproperties propertiesofofRes Res and haveinintensively investigated and reported on the MTA1-mediated properties of Res reported on theinin MTA1-mediated anticancer properties of Res and Pter inanticancer PCa in vitro and in vivo [3,4,and Pter PCa vitro [3,4,7,8,11–14,16]. Pter in PCa vitroand andininvivo vivo [3,4,7,8,11–14,16]. Pter in PCa in vitro and in vivo [3,4,7,8,11–14,16]. 7,8,11–14,16]. The Thepresent presentstudy studyaimed aimedtotoinvestigate investigatethe theanticancer anticancerefficacy efficacyand andMTA1 MTA1targeting targetingability abilityofof The present study aimed to investigate the anticancer efficacy and MTA1 targeting ability The present study aimed to investigate the anticancer MTA1 targeting ability of grape of grape powder extract (GPE) ininPCa lines. Grape powder extract consists ofofseven grape powder extract (GPE) PCacell cell lines. Grapeefficacy powderand extract consists sevenflavonoids, flavonoids, grape powder extract (GPE) in PCa cell lines. Grape powder extract consists of seven flavonoids, powder extract (GPE) in and PCa cell lines. Grape powder extract consists of seven flavonoids, including resveratrol anthocyanidins (Table 1)1)[37]. We cell-based assays including resveratrol andthree three anthocyanidins (Table [37]. Weperformed performed cell-basedincluding assayswith with including resveratrol and three anthocyanidins (Table 1) [37]. We performed cell-based assays with resveratrol and three anthocyanidins (Table 1) [37]. We performed cell-based assays with GPE GPE that GPE GPEinintwo twoPCa PCacell celllines linesusing usingRes Resand andPter Pteras asreference referencecompounds. compounds.Our Ourresults resultsindicate indicate thatin GPE GPE incell two PCaand cell lines Reseffects and Pter reference compounds. Ourindicate results indicate that GPE two PCa lines using Res using and Pter as reference compounds. Our results that GPE has has anticancer ininas PCa, while such Pter have the has anticancer and antimetastatic antimetastatic effects PCa, while stilbenes stilbenes such as as Res Res and and Pter have the has anticancer and antimetastatic effects in PCa, while stilbenes such as Res and Pter have the anticancer and antimetastatic effects in PCa, while stilbenes such as Res and Pter have the strongest strongest MTA1 inhibitory action. Therefore, grape extract enriched for stilbenes through unique strongest MTA1 inhibitory action. Therefore, grape extract enriched for stilbenes through unique strongest MTA1 inhibitory action. Therefore, grape extract enriched for stilbenes through unique MTA1 inhibitory action. Therefore, grape extract enriched for stilbenes through unique extraction extraction procedures may an dietary for and therapeutic extraction procedures mayrepresent represent aneffective effective dietaryagent agent forchemopreventive chemopreventive and therapeutic extraction procedures may represent an effective dietary agent for chemopreventive and therapeutic procedures may represent activity PCa. activityagainst against PCa. an effective dietary agent for chemopreventive and therapeutic activity activity against PCa. against PCa. Table Table1.1.Liquid LiquidChromatography Chromatographywith withtandem tandemmass massspectrometry spectrometry(LC-MS/MS) (LC-MS/MS)analysis analysisofofthe theGPE GPE Table 1. Liquid Chromatography with tandem mass spectrometry (LC-MS/MS) analysis of the GPE Table 1. Liquid Chromatography with tandem mass spectrometry (LC-MS/MS) analysis of the GPE (grape (grapepowder powderextract) extract)and andchemical chemicalstructures structuresofofits itscompounds. compounds. (grape powder extract) chemical structures its compounds. (grape powder extract) and and chemical structures of itsofcompounds.
Class Class Class
Compound
Compound ClassCompound Compound
Catechin Catechin Catechin Catechin
Chemical Structure
Chemical Structure Chemical ChemicalStructure Structure
Content Content(ppm) (ppm) Content (ppm) Content (ppm)
4014
4014 4014 4014
Epicatechin Epicatechin Epicatechin Epicatechin
1268
1268 1268 1268
Quercetin Quercetin Quercetin Quercetin
3429
3429 3429 3429
429
429 429 429
Phenols Phenols Phenols Phenols
Flavonols Flavonols Flavonols Flavonols Kaempferol Kaempferol KaempferolKaempferol
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Table 1. Cont. Nutrients 2018, 10, FOR PEER REVIEW Nutrients2018, 2018,10, 10,xxxFOR FORPEER PEERREVIEW REVIEW Nutrients Nutrients 2018, 2018,10, 10, FORPEER PEER REVIEW REVIEW Nutrients xxFOR Class Compound
Chemical Structure
3 of 12 of12 12 333of of12 12 3 of
Content (ppm)
Isorhamnetin Isorhamnetin Isorhamnetin Isorhamnetin Isorhamnetin Isorhamnetin
346
346 346 346 346 346
Taxifolin Taxifolin Taxifolin Taxifolin Taxifolin Taxifolin
656
656 656 656 656 656
88.5
88.5 88.5 88.5 88.5 88.5
Stilbenes Resveratrol Stilbenes Resveratrol Resveratrol Stilbenes Resveratrol StilbenesStilbenes Resveratrol Stilbenes Resveratrol
Cyanidin Cyanidin Cyanidin Cyanidin Cyanidin Cyanidin
508
508 508 508 508 508
Anthocyanins Peonidin Anthocyanins Peonidin Anthocyanins Peonidin Anthocyanins Peonidin Peonidin Anthocyanins Anthocyanins Peonidin
5034
5034 5034 5034 5034 5034
Malvidin Malvidin Malvidin Malvidin Malvidin Malvidin
2811
2811 2811 2811 2811 2811
2. Materials and Methods 2. Materials and Methods 2. Methods 2.Materials Materials and Methods 2. Materials andand Methods 2. and Methods Materials 2.1. Compounds 2.1. Compounds 2.1.2.1. Compounds 2.1.Compounds Compounds 2.1. Compounds Grape powder, which is aa proportional representation of the different varieties of table grapes Grape powder, which proportional representation of the different varieties of table grapes Grape powder, which representation of varieties of grapes Grape powder, which is aisis proportional representation of the different varieties of table grapes Grape powder, which isaaaproportional proportional representation ofthe thedifferent different varieties oftable table grapes Grape powder, which is proportional representation of the different varieties of table grapes grown in California, was obtained from the California Table Grape Commission. The grape powder grown in California, was obtained from the California Table Grape Commission. The grape powder grown in California, was obtained from the California Table Grape Commission. The grape powder grown in California, waswas obtained fromfrom the California Table Grape Commission. TheThe grape powder grown in California, California, was obtained from the California California Table Grape Commission. The grape powder grown in obtained the Table Grape Commission. powder extract (GPE) was prepared and standardized as described previously [37] and was aagrape generous gift extract (GPE) was prepared and standardized as described previously [37] and was generous gift extract (GPE) was prepared and standardized as described previously [37] and was a generous extract (GPE) was prepared and standardized as described previously [37] and was a generous giftgift extract (GPE) was prepared and standardized as described previously [37] and was a generous gift extract (GPE) was prepared and standardized as described previously [37] and was a generous gift from Dr. Richard van Breemen (Linus Pauling Institute, Oregon State University, Corvallis, OR, from Dr. Richard van Breemen (Linus Pauling Institute, Oregon State University, Corvallis, OR, from Dr. Richard van Breemen (Linus Pauling Institute, Oregon State University, Corvallis, OR, from Dr. Richard van Breemen (Linus Pauling Institute, Oregon State University, Corvallis, OR, USA). from Dr. Dr. Richard Richard van van Breemen Breemen (Linus (Linus Pauling Pauling Institute, Institute, Oregon Oregon State State University, University, Corvallis, Corvallis, OR, OR, from USA). Resveratrol and pterostilbene were purchased from Sigma-Aldrich (St. Louis, MO, USA) and USA). Resveratrol and pterostilbene were purchased from Sigma-Aldrich (St. Louis, MO, USA) and USA). Resveratrol and pterostilbene were purchased from Sigma-Aldrich (St. Louis, MO, USA) and Resveratrol and pterostilbene were purchased from Sigma-Aldrich (St. Louis, MO, USA) and were USA). Resveratrol and pterostilbene were purchased from Sigma-Aldrich (St. Louis, MO, USA) and USA).dissolved Resveratrol and pterostilbene were purchased from Sigma-Aldrich (St. Louis, MO, USA) and were in dimethyl sulfoxide (DMSO) for the in vitro experiments. were dissolved in dimethyl sulfoxide (DMSO) for the in vitro experiments. were dissolved in dimethyl sulfoxide (DMSO) for the in vitro experiments. dissolved in dimethyl sulfoxide (DMSO) for the in vitro experiments. were dissolved in dimethyl sulfoxide (DMSO) for the in vitro experiments. were dissolved in dimethyl sulfoxide (DMSO) for the in vitro experiments. 2.2. Cell Culture 2.2. Cell Culture 2.2. 2.2.Cell CellCulture Culture 2.2. Cell Culture Prostate cancer cells, DU145 and PC3M, were maintained in RPMI-1640 media containing 10 % Prostate cancer cells, DU145 and PC3M, were maintained in RPMI-1640 media containing 10 % Prostate Prostatecancer cancercells, cells,DU145 DU145and andPC3M, PC3M,were weremaintained maintainedin inRPMI-1640 RPMI-1640media mediacontaining containing10 10% % Prostate cancer cells, DU145 and PC3M, were maintained in RPMI-1640 media containing 10 % FBS in an incubator at 37 °C with 5% CO 2 as described previously [3,6,8–16,33,34,36]. Cells were FBS in an incubator at 37 °C with 5% CO as described previously [3,6,8–16,33,34,36]. Cells were FBS in an incubator at 37 °C with 5% CO 222as described previously [3,6,8–16,33,34,36]. Cells were FBS in an incubator at 37 °C with 5% CO as described previously [3,6,8–16,33,34,36]. Cells were FBS in an incubator at 37tandem °C with 5% CO 2 as described previously [3,6,8–16,33,34,36]. Cells were authenticated using short repeat profiling at Research Technology Support Facility, Michigan authenticated using short tandem repeat profiling at Research Technology Support Facility, Michigan authenticated authenticatedusing usingshort shorttandem tandemrepeat repeatprofiling profilingat atResearch ResearchTechnology TechnologySupport SupportFacility, Facility,Michigan Michigan authenticated using short tandem repeat profiling at Research Technology Support Facility, Michigan State University. State University. State University. State University. University. State
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2.2. Cell Culture Prostate cancer cells, DU145 and PC3M, were maintained in RPMI-1640 media containing 10% FBS in an incubator at 37 ◦ C with 5% CO2 as described previously [3,6,8–16,33,34,36]. Cells were authenticated using short tandem repeat profiling at Research Technology Support Facility, Michigan State University. 2.3. MTT Assay Cell viability of DU145 and PC3M cells was measured after treatment with GPE (2–200 µg/mL), Res (5–100 µM), and Pter (5–100 µM) using MTT assay (Sigma-Aldrich, St. Louis, MO, USA) as described previously [6,16]. Briefly, the cells were seeded in 96-well plates and treated with vehicle, GPE, Res, or Pter. Absorbance of the formazan was measured using BioTek Synergy-4 plate reader (BioTek, Winooski, VT, USA) after 72 h of treatment. The % cell viability was calculated assuming 100% viability in vehicle-treated (control) wells. 2.4. Proliferation Assay DU145 and PC3M cells (2 × 103 ) were seeded in a 35-mm cell culture dish. The media with appropriate compound (GPE, Res, or Pter) was changed every other day. The proliferation of the cells was determined by counting the cells every other day over a period of 10 days. 2.5. Colony Formation Assay Colony formation assay was performed as described previously [33]. Briefly, cells (5 × 103 ) were seeded in a 35 mm cell culture dish for a 21-day observation time. The media with appropriate compound was changed every other day. When colonies were freely visible (>50 cells/colony) in vehicle-treated dish, cells were fixed with formaldehyde and stained with 0.1% crystal violet solution. Colonies were visualized by imaging each dish using Amersham Imager 600 (GE Healthcare Bio-Sciences, Pittsburg, PA, USA). ImageQuant TL software (GE Healthcare Bio-Sciences, Pittsburg, PA, USA) was used for counting the number of colonies in each dish. 2.6. Wound-Healing Assay Wound-healing assay was performed as described previously [33,36]. Briefly, 95% confluent cells seeded in 6-well plates were starved in low serum media (0.1% serum) overnight, after which three separate wounds were scratched across the well. The media with appropriate compound was changed every other day. The wound was imaged daily until the wounds of vehicle-treated (control) cells were completely closed using the EVOS XL Core microscope (ThermoFisher Scientific, Waltham, MA, USA). Wound area was calculated using the ImageJ software (NIH, Bethesda, MD, USA). % wound area was quantitated assuming 100% for vehicle-treated cells at 0 h. 2.7. Western Blot Western blot analysis was carried out as previously described [33,36]. Briefly, cells were treated with various concentrations of GPE (25–200 µg/mL), Res (50 µM), or Pter (50 µM) for 24 h, and total protein was extracted. Protein concentration was measured using Bio-Rad protein assay reagent (Bio-Rad Laboratories, Hercules, CA, USA). An equal amount of protein was resolved in 10–15% gels and transferred to a polyvinylidene difluoride (PVDF) membrane. After blocking the membranes for non-specificity, they were probed with MTA1 (1:2000), p21 (1:1000), cleaved caspase 3 (1:1000), PTEN (1:1000), Cyclin D1 (1:1000), and pAkt (1:1000) (Cell Signaling Technology, Danvers, MA, USA) primary antibodies. β-actin antibody (1:2500) (Santa Cruz, Dallas, TX, USA) was used as a loading control. Signals were visualized using enhanced chemoluminescence. Densitometry was performed using Image J software (NIH, Bethesda, MD, USA).
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2.8. Statistical Analysis The differences between the groups were analyzed by one-way analysis of variance (ANOVA). All statistics were performed using GraphPad Prism 7 software (GraphPad Software, La Jolla, CA, USA). The statistical significance was set as p < 0.05. All data are cumulative of at least three Nutrients 2018, 10, x FOR PEER REVIEW 5 of 12 independent experiments.
Results 3.3. Results Thecytotoxic cytotoxiceffects effectsof ofGPE GPEwere wereevaluated evaluatedand andcompared comparedto tothose thoseof ofRes Resand andPter Pterby byMTT MTTcell cell The viability assay in DU145 and PC3M aggressive prostate cancer cell lines. The cells were treated with viability assay in DU145 and PC3M aggressive prostate cancer cell lines. The cells were treated with variousconcentrations concentrationsofof GPE (5–200 μg/mL), Res (5–100 or 1.14–22.8 μg/mL), (5–100 various GPE (5–200 µg/mL), Res (5–100 µMμM or 1.14–22.8 µg/mL), andand Pter Pter (5–100 µM μM or 1.28–25.6 μg/mL) for 72 h. As shown in Figure 1A, treatment with GPE had a modest cytotoxic or 1.28–25.6 µg/mL) for 72 h. As shown in Figure 1A, treatment with GPE had a modest cytotoxic effecteven evenatathigh high μg/mL dose, whereas Pter significantly inhibited the DU145 and effect 200200 µg/mL dose, whereas Res Res and and Pter significantly inhibited the DU145 and PC3M PC3M cells’ viability in relatively low concentrations. IC50 of values of Res and Pter in DU145 and cells’ viability in relatively low concentrations. The IC50The values Res and Pter in DU145 and PC3M PC3M cells were in accordance with our previous reports [10], while GPE showed very low activity cells were in accordance with our previous reports [10], while GPE showed very low activity in both in both lines, IC50 of 107inμg/mL DU145 cells.not Weable were able to IC determine IC50 cell lines,cell with IC50with values ofvalues 107 µg/mL DU145in cells. We were tonot determine 50 values for values for (5–200 GPE doses (5–200 μg/mL) used in PC3M cells.investigate To furtherthe investigate inhibition of cell GPE doses µg/mL) used in PC3M cells. To further inhibitionthe of cell proliferation proliferation by GPE, we performed cell-counting assay upon treatment with compounds. We by GPE, we performed cell-counting assay upon treatment with compounds. We counted cell numbers counted cellday numbers every other daythat forGPE 10 days and found that GPE every other for 10 days and found significantly inhibited cell significantly proliferation inhibited comparedcell to proliferation compared to control untreated cells in all concentrations tested in both DU145 and control untreated cells in all concentrations tested in both DU145 and PC3M cells (Figure 1B). In DU145 PC3M cells concentrations (Figure 1B). InofDU145 cells, concentrations of GPE (15 and 100 μg/mL) were cells, higher GPE (15 andhigher 100 µg/mL) were significantly more potent at inhibiting significantly potent inhibiting < 0.01; < 0.0001) than Resatbut notdoses Pter. In cell growth (pmore < 0.01; p < at 0.0001) than cell Resgrowth but not(pPter. InpPC3M cells, GPE high (15PC3M and cells, GPE at high doses (15 and 100 μg/mL) as well as Res and Pter showed significant differences 100 µg/mL) as well as Res and Pter showed significant differences compared to control vehicle-treated compared to control vehicle-treated cells (p < 0.001; p < 0.0001). cells (p < 0.001; p < 0.0001).
Figure Figure1.1. Effects Effectsof ofGPE GPEon oncell cellviability viabilityand andcell cellproliferation. proliferation. (A) (A)Cell Cellviability viabilityanalysis analysisof ofDU145 DU145 (left) and PC3M (right) prostate cancer cells treated with GPE (2–200 µg/mL), Res (1.14–22.8 (left) and PC3M (right) prostate cancer cells treated with GPE (2–200 μg/mL), Res (1.14–22.8µg/mL), μg/mL), and andPter Pter(1.28–25.6 (1.28–25.6µg/mL). μg/mL). Data Data represent representthe themean mean±± scanning scanning electron electron microscopy microscopy (SEM) (SEM) of ofthree three independent sets of experiments. (B) Proliferation assay of DU145 (left) and PC3M (right) cells independent sets of experiments. (B) Proliferation assay of DU145 (left) and PC3M (right) cellsafter after treatment with GPE (1.5; 15; 100 µg/mL) and Res and Pter (1.5 µg/mL) for 10 days. Data represent treatment with GPE (1.5; 15; 100 μg/mL) and Res and Pter (1.5 μg/mL) for 10 days. Data represent the the mean ± SEM of three independent sets of experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, and mean ± SEM of three independent sets of experiments. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < **** p < 0.0001 (one-way analysis of variance (ANOVA)) were assessed as significant differences between 0.0001 (one-way analysis of variance (ANOVA)) were assessed as significant differences between treated and Ctrl vehicle cells. treated and Ctrl vehicle cells.
To characterize the anti-metastatic capacity of GPE, we examined the effect of GPE on the colony formation and wound healing in DU145 and PC3M cells. As shown in Figure 2A, colony-forming ability was decreased in DU145 cells, with an increased GPE concentration gradient reaching significance at 15 μg/mL (p < 0.001) and 100 μg/mL (p < 0.0001). In more aggressive PC3M cells, GPE
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To characterize the anti-metastatic capacity of GPE, we examined the effect of GPE on the colony formation and wound healing in DU145 and PC3M cells. As shown in Figure 2A, colony-forming ability was decreased in DU145 cells, with an increased GPE concentration gradient reaching significance at 15 µg/mL (p < 0.001) and 100 µg/mL (p < 0.0001). In more aggressive PC3M cells, GPE treatment at only a high (100 µg/mL) dose caused a significant reduction of colony-forming ability (p < 0.0001) Nutrients x FOR PEER REVIEW 6 of of 12 (Figure 2018, 2B). 10, However, these results indicated that only the effects of the highest concentration GPE (100 µg/mL) were comparable with the effects of Res and Pter alone at a relatively low dose relatively low After dose (1.5 μg/mL). After this, a assay wound-healing assay was performed in order toeffects measure (1.5 µg/mL). this, a wound-healing was performed in order to measure the of the effects of GPE treatment on tumor cell migration. The migration of DU145 and PC3M cells was GPE treatment on tumor cell migration. The migration of DU145 and PC3M cells was decreased decreased with anGPE increased GPE concentration gradient (Figure 3A,B).inAs shown Figure 3A, with an increased concentration gradient (Figure 3A,B). As shown Figure 3A,inreduction in reduction in the cell migration of GPE-treated DU145 cells was highly significant (p < 0.001) in a dosethe cell migration of GPE-treated DU145 cells was highly significant (p < 0.001) in a dose-dependent dependent manner, to compared to control vehicle-treated cells. Interestingly, GPE-treated PC3M cells, manner, compared control vehicle-treated cells. Interestingly, GPE-treated PC3M cells, which are which are characterized as more than aggressive cells, were affected in their migration characterized as more aggressive DU145 than cells, DU145 were affected in their migration capabilities by capabilities by GPE treatment stronger than DU145 cells. These results indicated that GPE could GPE treatment stronger than DU145 cells. These results indicated that GPE could reduce migration reduce migration of aggressive prostate cancer cells in manner a dose-dependent at asdose. low of aggressive prostate cancer cells in a dose-dependent starting at manner as low asstarting 1.5 µg/mL as 1.5again, μg/mL dose. again, Res strong and Pter alsotoshowed strong ability to reduce migration of Once Res and Once Pter also showed ability reduce migration of prostate cancer cells at 1.5 prostate cancer cells at 1.5 μg/mL dose. µg/mL dose.
Figure 2. 2. GPE GPE reduces reduces colony colony formation formation in in DU145 Figure DU145 and and PC3M PC3M prostate prostate cancer cancer cells. cells. Representative Representative images of colony formation ability of (A) DU145 and (B) PC3M cells after treatment with GPE GPE (1.5; (1.5; images of colony formation ability of (A) DU145 and (B) PC3M cells after treatment with 15; 100 µg/mL) and Res and Pter (1.5 µg/mL). Data represent the mean ± SEM of three independent 15; 100 μg/mL) and Res and Pter (1.5 μg/mL). Data represent the mean ± SEM of three independent experiments with duplicate wells. *** p < 0.001 and **** p < 0.0001 (one-way ANOVA) were assessed as experiments with duplicate wells. ***p < 0.001 and ****p < 0.0001 (one-way ANOVA) were assessed as significant differences between treated and Ctrl vehicle cells. significant differences between treated and Ctrl vehicle cells.
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Figure Figure3.3.GPE GPEreduces reducesmigration migrationof ofDU145 DU145and andPC3M PC3Mprostate prostatecancer cancercells. cells.Representative Representativeimages imagesof of migration ability of (A) DU145 and (B) PC3M cells after treatment with GPE (1.5; 15; 100 µg/mL) migration ability of (A) DU145 and (B) PC3M cells after treatment with GPE (1.5; 15; 100 μg/mL)and and Res Resand andPter Pter(1.5 (1.5µg/mL). μg/mL). Right: Right: quantitation quantitation of of wound wound widths, widths, as as % % wound wound area area isis shown shownfor foreach each cell line. Data represent the mean ± SEM of six separate wounds and three independent experiments. cell line. Data represent the mean ± SEM of six separate wounds and three independent experiments. *** p
